Heterotandem bicyclic peptide complex

ABSTRACT

The present invention relates to a heterotandem bicyclic peptide complex which comprises a first peptide ligand, which binds to EphA2, conjugated via a linker to two second peptide ligands, which bind to CD137. The invention also relates to the use of said heterotandem bicyclic peptide complex in preventing, suppressing or treating cancer.

FIELD OF THE INVENTION

The present invention relates to a heterotandem bicyclic peptide complexwhich comprises a first peptide ligand, which binds to EphA2, conjugatedvia a linker to two second peptide ligands, which bind to CD137. Theinvention also relates to the use of said heterotandem bicyclic peptidecomplex in preventing, suppressing or treating cancer.

BACKGROUND OF THE INVENTION

Cyclic peptides can bind with high affinity and target specificity toprotein targets and hence are an attractive molecule class for thedevelopment of therapeutics. In fact, several cyclic peptides arealready successfully used in the clinic, as for example theantibacterial peptide vancomycin, the immunosuppressant drugcyclosporine or the anti-cancer drug octreotide (Driggers et al. (2008),Nat Rev Drug Discov 7 (7), 608-24). Good binding properties result froma relatively large interaction surface formed between the peptide andthe target as well as the reduced conformational flexibility of thecyclic structures. Typically, macrocycles bind to surfaces of severalhundred square angstrom, as for example the cyclic peptide CXCR4antagonist CVX15 (400 Å²; Wu et al. (2007), Science 330, 1066-71), acyclic peptide with the Arg-Gly-Asp motif binding to integrin αVb3 (355Å²) (Xiong et al. (2002), Science 296 (5565), 151-5) or the cyclicpeptide inhibitor upain-1 binding to urokinase-type plasminogenactivator (603 Å²; Zhao et al. (2007), J Struct Biol 160 (1), 1-10).

Due to their cyclic configuration, peptide macrocycles are less flexiblethan linear peptides, leading to a smaller loss of entropy upon bindingto targets and resulting in a higher binding affinity. The reducedflexibility also leads to locking target-specific conformations,increasing binding specificity compared to linear peptides. This effecthas been exemplified by a potent and selective inhibitor of matrixmetalloproteinase 8 (MMP-8) which lost its selectivity over other MMPswhen its ring was opened (Cherney et al. (1998), J Med Chem 41 (11),1749-51). The favorable binding properties achieved throughmacrocyclization are even more pronounced in multicyclic peptides havingmore than one peptide ring as for example in vancomycin, nisin andactinomycin.

Different research teams have previously tethered polypeptides withcysteine residues to a synthetic molecular structure (Kemp and McNamara(1985), J. Org. Chem; Timmerman et al. (2005), ChemBioChem). Meloen andco-workers had used tris(bromomethyl)benzene and related molecules forrapid and quantitative cyclisation of multiple peptide loops ontosynthetic scaffolds for structural mimicry of protein surfaces(Timmerman et al. (2005), ChemBioChem).

Methods for the generation of candidate drug compounds wherein saidcompounds are generated by linking cysteine containing polypeptides to amolecular scaffold as for example1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA) aredisclosed in WO 2019/122860 and WO 2019/122863.

Phage display-based combinatorial approaches have been developed togenerate and screen large libraries of bicyclic peptides to targets ofinterest (Heinis et al. (2009), Nat Chem Biol 5 (7), 502-7 and WO2009/098450). Briefly, combinatorial libraries of linear peptidescontaining three cysteine residues and two regions of six random aminoacids (Cys-(Xaa)₆-Cys-(Xaa)₆-Cys) were displayed on phage and cyclisedby covalently linking the cysteine side chains to a small molecule(tris-(bromomethyl)benzene).

SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided aheterotandem bicyclic peptide complex comprising:

-   -   (a) a first peptide ligand which binds to EphA2 and which has        the sequence A-[HArg]-D-C_(i)[HyP]LVNPLC_(ii)LEP[d1Nal]WTC_(iii)        (SEQ ID NO: 1; BCY13118); conjugated via an        N-(acid-PEG₃)-N-bis(PEG₃-azide) linker to    -   (b) two second peptide ligands which bind to CD137 both of which        have the sequence        Ac—C_(i)[tBuAla]PE[D-Lys(PYA)]PYC_(ii)FADPY[Nle]C_(iii)-A (SEQ        ID NO: 2; BCY8928);        wherein each of said peptide ligands comprise a polypeptide        comprising three reactive cysteine groups (C_(i), C_(ii) and        C_(iii)), separated by two loop sequences, and a molecular        scaffold which is        1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA)        and which forms covalent bonds with the reactive cysteine groups        of the polypeptide such that two polypeptide loops are formed on        the molecular scaffold;        wherein Ac represents acetyl, HArg represents homoarginine, HyP        represents trans-4-hydroxy-L-proline, d1Nal represents        D-1-naphthylalanine, tBuAla represents t-butyl-alanine, PYA        represents 4-pentynoic acid and Nle represents norleucine.

According to a further aspect of the invention, there is provided apharmaceutical composition comprising a heterotandem bicyclic peptidecomplex as defined herein in combination with one or morepharmaceutically acceptable excipients.

According to a further aspect of the invention, there is provided aheterotandem bicyclic peptide complex as defined herein for use inpreventing, suppressing or treating cancer.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Analysis of the EphA2/CD137 heterotandem bicyclic peptidecomplex BCY13272 in the Promega CD137 luciferase reporter assay in thepresence of EphA2 expressing A549, PC-3 and HT29 cells (n=3). BCY13626is a heterotandem bicyclic peptide complex similar to BCY13272 butcomprises D-amino acids and does not bind to EphA2 or CD137.

FIG. 2: Plasma concentration versus time plot of BCY13272 from a 5.5mg/kg IV dose in CD1 mice (n=3), a 3.6 mg/kg IV infusion (15 min) in SDrats (n=3) and a 8.9 mg/kg IV infusion (15 min) in cynomolgus monkeys(n=2). The pharmacokinetic profile of BCY13272 has a terminal half-lifeof 2.9 hours in CD-1 mice, 2.5 hours in SD Rats and 8.9 hours in cyno.

FIG. 3: Anti-tumor activity of BCY13272 in a syngeneic MC38 tumor model.(A) MC38 tumor volumes during and after BCY13272 treatment. Number ofcomplete responder (CR) mice on D28 (and that remain CRs on D62) areindicated in parentheses. BIW: twice weekly dosing; IV: intravenousadministration. (B) Tumor growth curves of complete responder animals toBCY13272 and naïve age-matched control animals after MC38 tumor cellimplantation. CR: complete responder.

FIG. 4: BCY13272 induces IFN-γ cytokine secretion in a (A) PBMC/MC38 anda (B) PBMC/HT29 co-culture assay. BCY12762 is a heterotandem bicyclicpeptide complex that binds to EphA2 but does not bind to CD137. BCY13692is a heterotandem bicycle peptide complex that binds to CD137 but doesnot bind to EphA2. (C) Plot of EC50 (nM) values of BCY13272 induced IL-2and IFN-γ secretion in PBMC coculture assay with MC38 (mouse) cell linewith 5 PBMC donors and HT1080 (human) cell line with 4 PBMC donors.

FIG. 5: Surface plasmon resonance (SPR) binding of BCY13272 toimmobilized (A) EphA2 and (B) CD137.

FIG. 6: Formula of BCY13272.

FIG. 7: Formula of BCY14414.

FIG. 8: Formula of BCY14417.

FIG. 9: Formula of BCY14418.

FIG. 10: Formula of BCY15217.

FIG. 11: Formula of BCY15218.

DETAILED DESCRIPTION OF THE INVENTION

According to a first aspect of the invention, there is provided aheterotandem bicyclic peptide complex comprising:

-   -   (a) a first peptide ligand which binds to EphA2 and which has        the sequence A-[HArg]-D-C_(i)[HyP]LVNPLC_(ii)LEP[d1Nal]WTC_(iii)        (SEQ ID NO: 1; BCY13118); conjugated via an        N-(acid-PEG₃)-N-bis(PEG₃-azide) linker to    -   (b) two second peptide ligands which bind to CD137 both of which        have the sequence        Ac—C_(i)[tBuAla]PE[D-Lys(PYA)]PYC_(ii)FADPY[Nle]C_(iii)-A (SEQ        ID NO: 2; BCY8928);        wherein each of said peptide ligands comprise a polypeptide        comprising three reactive cysteine groups (C_(i), C_(ii) and        C_(iii)), separated by two loop sequences, and a molecular        scaffold which is        1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA)        and which forms covalent bonds with the reactive cysteine groups        of the polypeptide such that two polypeptide loops are formed on        the molecular scaffold;        wherein Ac represents acetyl, HArg represents homoarginine, HyP        represents trans-4-hydroxy-L-proline, d1Nal represents        D-1-naphthylalanine, tBuAla represents t-butyl-alanine, PYA        represents 4-pentynoic acid and Nle represents norleucine.

References herein to a N-(acid-PEG₃)-N-bis(PEG₃-azide) linker include:

In one embodiment, the heterotandem bicyclic peptide complex is BCY13272(structure shown in FIG. 6).

Full details of BCY13272 are shown in Table A below:

TABLE A Composition of BCY13272 Complex EphA2 Attachment CD137Attachment No. BCY No. Point Linker BCY No. Point BCY13272 BCY13118N-terminus N-(acid-PEG₃)- BCY8928, dLys (PYA)4 N-bis(PEG₃-azide) BCY8928

Data is presented here in FIG. 3 which demonstrates that BCY13272 leadsto a significant anti tumor effect in a MC38 tumor model in mice.

Reference herein is made to certain analogues (i.e. modifiedderivatives) and metabolites of BCY13272, each of which form additionalaspects of the invention and are summarised in Table B below:

TABLE B Composition of labelled analoques and potential metabolites ofBCY13272 Complex EphA2 Attachment CD137 Attachment No. BCY No. PointLinker BCY No. Point Modifier BCY14414 BCY13118 N- N-(acid-PEG₃)-N-BCY8928 dLys(PYA)4 N/A terminus bis(PEG₃-azide) BCY13389 dLys(PYA)4BCY14417 BCY13118 N- N-(acid-PEG₃)-N- BCY8928 dLys(PYA)4 Peg12-Biotinterminus bis(PEG₃-azide) BCY13389 dLys(PYA)4 BCY14418 BCY13118 N-N-(acid-PEG₃)-N- BCY8928 dLys(PYA)4 Alexa terminus bis(PEG₃-azide)BCY13389 dLys(PYA)4 Fluor ® 488 BCY15217 BCY13118 N- N-(acid-PEG₃)-N-BCY14601 dLys(PYA)4 N/A terminus bis(PEG₃-azide) BCY14601 dLys(PYA)4BCY15218 BCY13118 N- N-(acid-PEG₃)-N- BCY8928 dLys(PYA)4 N/A terminusbis(PEG₃-azide) BCY14601 dLys(PYA)4wherein BCY14601 represents a bicyclic peptide ligand having thesequence of C_(i)[tBuAla]PE[D-Lys(PYA)]PYC_(ii)FADPY[Nle]C_(iii)-A (SEQID NO: 3) with TATA as a molecular scaffold;and wherein BCY13389 represents a bicyclic peptide ligand having thesequence of [Ac]C_(i)[tBuAla]PE[D-Lys(PYA)]PYC_(ii)FADPY[Nle]C_(iii)-K(SEQ ID NO: 4) with TATA as a molecular scaffold.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art, such as in the arts of peptide chemistry, cell culture andphage display, nucleic acid chemistry and biochemistry. Standardtechniques are used for molecular biology, genetic and biochemicalmethods (see Sambrook et al., Molecular Cloning: A Laboratory Manual,3rd ed., 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.; Ausubel et al., Short Protocols in Molecular Biology (1999) 4^(th)ed., John Wiley & Sons, Inc.), which are incorporated herein byreference.

Nomenclature

Numbering

When referring to amino acid residue positions within compounds of theinvention, cysteine residues (C_(i), C_(ii) and C_(iii)) are omittedfrom the numbering as they are invariant, therefore, the numbering ofamino acid residues within SEQ ID NO: 1 is referred to as below:

(SEQ ID NO: 1)C_(i)HyP₁-L₂-V₃-N₄-P₅-L₆-C_(ii)-L₇-E₈-P₉-d1Nal₁₀-W₁₁-T₁₂-C_(iii).

For the purpose of this description, the bicyclic peptides are cyclisedwith 1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA) andyield a tri-substituted structure. Cyclisation with TATA occurs onC_(i), C_(ii), and C_(iii).

Molecular Format

N- or C-terminal extensions to the bicycle core sequence are added tothe left or right side of the sequence, separated by a hyphen. Forexample, an N-terminal βAla-Sar10-Ala tail would be denoted as:

(SEQ ID NO: X) βAla-Sar10-A.Inversed Peptide Sequences

In light of the disclosure in Nair et al (2003) J Immunol 170(3),1362-1373, it is envisaged that the peptide sequences disclosed hereinwould also find utility in their retro-inverso form. For example, thesequence is reversed (i.e. N-terminus becomes C-terminus and vice versa)and their stereochemistry is likewise also reversed (i.e. D-amino acidsbecome L-amino acids and vice versa). For the avoidance of doubt,references to amino acids either as their full name or as their aminoacid single or three letter codes are intended to be represented hereinas L-amino acids unless otherwise stated. If such an amino acid isintended to be represented as a D-amino acid then the amino acid will beprefaced with a lower case d within square parentheses, for example[dA], [dD], [dE], [dK], [d1Nal], [dNle], etc.

Advantages of the Peptide Ligands

Certain heterotandem bicyclic peptide complexes of the present inventionhave a number of advantageous properties which enable them to beconsidered as suitable drug-like molecules for injection, inhalation,nasal, ocular, oral or topical administration. Such advantageousproperties include:

-   -   Species cross-reactivity. This is a typical requirement for        preclinical pharmacodynamics and pharmacokinetic evaluation;    -   Protease stability. Heterotandem bicyclic peptide complexes        should ideally demonstrate stability to plasma proteases,        epithelial (“membrane-anchored”) proteases, gastric and        intestinal proteases, lung surface proteases, intracellular        proteases and the like. Protease stability should be maintained        between different species such that a heterotandem bicyclic        peptide lead candidate can be developed in animal models as well        as administered with confidence to humans;    -   Desirable solubility profile. This is a function of the        proportion of charged and hydrophilic versus hydrophobic        residues and intra/inter-molecular H-bonding, which is important        for formulation and absorption purposes;    -   Selectivity. Certain heterotandem bicyclic peptide complexes of        the invention demonstrate good selectivity over other targets;    -   An optimal plasma half-life in the circulation. Depending upon        the clinical indication and treatment regimen, it may be        required to develop a heterotandem bicyclic peptide complex for        short exposure in an acute illness management setting, or        develop a heterotandem bicyclic peptide complex with enhanced        retention in the circulation, and is therefore optimal for the        management of more chronic disease states. Other factors driving        the desirable plasma half-life are requirements of sustained        exposure for maximal therapeutic efficiency versus the        accompanying toxicology due to sustained exposure of the agent.    -   Crucially, data is presented herein where the heterotandem        bicyclic peptide complex of the invention demonstrates        anti-tumor efficacy when dosed at a frequency that does not        maintain plasma concentrations above the in vitro EC₅₀ of the        compound. This is in contrast to larger recombinant biologic        (i.e. antibody based) approaches to CD137 agonism or bispecific        CD137 agonism (Segal et al., Clin Cancer Res., 23(8):1929-1936        (2017), Claus et al., Sci Trans Med., 11(496): eaav5989, 1-12        (2019), Hinner et al., Clin Cancer Res., 25(19):5878-5889        (2019)). Without being bound by theory, the reason for this        observation is thought to be due to the fact that heterotandem        bicycle complexes have relatively low molecular weight        (typically <15 kDa), they are fully synthetic and they are tumor        targeted agonists of CD137. As such, they have relatively short        plasma half lives but good tumor penetrance and retention. Data        is presented herein which fully supports these advantages. For        example, anti-tumor efficacy in syngeneic rodent models in mice        with humanized CD137 is demonstrated either daily or every        3^(rd) day. In addition, intraperitoneal pharmacokinetic data        shows that the plasma half life is <3 hours, which would predict        that the circulating concentration of the complex would        consistently drop below the in vitro EC₅₀ between doses.        Furthermore, tumor pharmacokinetic data shows that levels of        heterotandem bicycle complex in tumor tissue may be higher and        more sustained as compared to plasma levels.    -   It will be appreciated that this observation forms an important        further aspect of the invention. Thus, according to a further        aspect of the invention, there is provided a method of treating        cancer which comprises administration of a heterotandem bicyclic        peptide complex as defined herein at a dosage frequency which        does not sustain plasma concentrations of said complex above the        in vitro EC₅₀ of said complex.    -   Immune Memory. Coupling the cancer cell binding bicyclic peptide        ligand with the immune cell binding bicyclic peptide ligand        provides the synergistic advantage of immune memory. The        heterotandem bicyclic peptide complex of the invention is        believed not only to eradicate tumors but upon readministration        of the tumorigenic agent, none of the inoculated complete        responder mice developed tumors. This indicates that treatment        with the heterotandem bicyclic peptide complex of the invention        has induced immunogenic memory in the complete responder mice.        This has a significant clinical advantage in order to prevent        recurrence of said tumor once it has been initially controlled        and eradicated.        Peptide Ligands

A peptide ligand, as referred to herein, refers to a peptide covalentlybound to a molecular scaffold. Typically, such peptides comprise two ormore reactive groups (i.e. cysteine residues) which are capable offorming covalent bonds to the scaffold, and a sequence subtended betweensaid reactive groups which is referred to as the loop sequence, since itforms a loop when the peptide is bound to the scaffold. In the presentcase, the peptides comprise at least three reactive groups selected fromcysteine, 3-mercaptopropionic acid and/or cysteamine and form at leasttwo loops on the scaffold.

Pharmaceutically Acceptable Salts

It will be appreciated that salt forms are within the scope of thisinvention, and references to peptide ligands include the salt forms ofsaid ligands.

The salts of the present invention can be synthesized from the parentcompound that contains a basic or acidic moiety by conventional chemicalmethods such as methods described in Pharmaceutical Salts: Properties,Selection, and Use, P. Heinrich Stahl (Editor), Camille G. Wermuth(Editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002.Generally, such salts can be prepared by reacting the free acid or baseforms of these compounds with the appropriate base or acid in water orin an organic solvent, or in a mixture of the two.

Acid addition salts (mono- or di-salts) may be formed with a widevariety of acids, both inorganic and organic. Examples of acid additionsalts include mono- or di-salts formed with an acid selected from thegroup consisting of acetic, 2,2-dichloroacetic, adipic, alginic,ascorbic (e.g. L-ascorbic), L-aspartic, benzenesulfonic, benzoic,4-acetamidobenzoic, butanoic, (+) camphoric, camphor-sulfonic,(+)-(1S)-camphor-10-sulfonic, capric, caproic, caprylic, cinnamic,citric, cyclamic, dodecylsulfuric, ethane-1,2-disulfonic,ethanesulfonic, 2-hydroxyethanesulfonic, formic, fumaric, galactaric,gentisic, glucoheptonic, D-gluconic, glucuronic (e.g. D-glucuronic),glutamic (e.g. L-glutamic), α-oxoglutaric, glycolic, hippuric,hydrohalic acids (e.g. hydrobromic, hydrochloric, hydriodic),isethionic, lactic (e.g. (+)-L-lactic, (±)-DL-lactic), lactobionic,maleic, malic, (−)-L-malic, malonic, (±)-DL-mandelic, methanesulfonic,naphthalene-2-sulfonic, naphthalene-1,5-disulfonic,1-hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic,palmitic, pamoic, phosphoric, propionic, pyruvic, L-pyroglutamic,salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulfuric,tannic, (+)-L-tartaric, thiocyanic, p-toluenesulfonic, undecylenic andvaleric acids, as well as acylated amino acids and cation exchangeresins.

One particular group of salts consists of salts formed from acetic,hydrochloric, hydriodic, phosphoric, nitric, sulfuric, citric, lactic,succinic, maleic, malic, isethionic, fumaric, benzenesulfonic,toluenesulfonic, sulfuric, methanesulfonic (mesylate), ethanesulfonic,naphthalenesulfonic, valeric, propanoic, butanoic, malonic, glucuronicand lactobionic acids. One particular salt is the hydrochloride salt.Another particular salt is the acetate salt.

If the compound is anionic, or has a functional group which may beanionic (e.g., —COOH may be —COO⁻), then a salt may be formed with anorganic or inorganic base, generating a suitable cation. Examples ofsuitable inorganic cations include, but are not limited to, alkali metalions such as Li⁺, Na⁺ and K⁺, alkaline earth metal cations such as Ca²⁺and Mg²⁺, and other cations such as Al³⁺ or Zn⁺. Examples of suitableorganic cations include, but are not limited to, ammonium ion (i.e., NH₄⁺) and substituted ammonium ions (e.g., NH₃R⁺, NH₂R₂ ⁺, NHR₃ ⁺, NR₄ ⁺).Examples of some suitable substituted ammonium ions are those derivedfrom: methylamine, ethylamine, diethylamine, propylamine,dicyclohexylamine, triethylamine, butylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine, benzylamine,phenylbenzylamine, choline, meglumine, and tromethamine, as well asamino acids, such as lysine and arginine. An example of a commonquaternary ammonium ion is N(CH₃)₄ ⁺.

Where the compounds of the invention contain an amine function, thesemay form quaternary ammonium salts, for example by reaction with analkylating agent according to methods well known to the skilled person.Such quaternary ammonium compounds are within the scope of theinvention.

Modified Derivatives

It will be appreciated that modified derivatives of the peptide ligandsas defined herein are within the scope of the present invention.Examples of such suitable modified derivatives include one or moremodifications selected from: N-terminal and/or C-terminal modifications;replacement of one or more amino acid residues with one or morenon-natural amino acid residues (such as replacement of one or morepolar amino acid residues with one or more isosteric or isoelectronicamino acids; replacement of one or more non-polar amino acid residueswith other non-natural isosteric or isoelectronic amino acids); additionof a spacer group; replacement of one or more oxidation sensitive aminoacid residues with one or more oxidation resistant amino acid residues;replacement of one or more amino acid residues with an alanine,replacement of one or more L-amino acid residues with one or moreD-amino acid residues; N-alkylation of one or more amide bonds withinthe bicyclic peptide ligand; replacement of one or more peptide bondswith a surrogate bond; peptide backbone length modification;substitution of the hydrogen on the alpha-carbon of one or more aminoacid residues with another chemical group, modification of amino acidssuch as cysteine, lysine, glutamate/aspartate and tyrosine with suitableamine, thiol, carboxylic acid and phenol-reactive reagents so as tofunctionalise said amino acids, and introduction or replacement of aminoacids that introduce orthogonal reactivities that are suitable forfunctionalisation, for example azide or alkyne-group bearing amino acidsthat allow functionalisation with alkyne or azide-bearing moieties,respectively.

In one embodiment, the modified derivative comprises an N-terminaland/or C-terminal modification. In a further embodiment, wherein themodified derivative comprises an N-terminal modification using suitableamino-reactive chemistry, and/or C-terminal modification using suitablecarboxy-reactive chemistry. In a further embodiment, said N-terminal orC-terminal modification comprises addition of an effector group,including but not limited to a cytotoxic agent, a radiochelator or achromophore.

In a further embodiment, the modified derivative comprises an N-terminalmodification. In a further embodiment, the N-terminal modificationcomprises an N-terminal acetyl group. In this embodiment, the N-terminalcysteine group (the group referred to herein as C_(i)) is capped withacetic anhydride or other appropriate reagents during peptide synthesisleading to a molecule which is N-terminally acetylated. This embodimentprovides the advantage of removing a potential recognition point foraminopeptidases and avoids the potential for degradation of the bicyclicpeptide.

In an alternative embodiment, the N-terminal modification comprises theaddition of a molecular spacer group which facilitates the conjugationof effector groups and retention of potency of the bicyclic peptide toits target.

In a further embodiment, the modified derivative comprises a C-terminalmodification. In a further embodiment, the C-terminal modificationcomprises an amide group. In this embodiment, the C-terminal cysteinegroup (the group referred to herein as C_(iii)) is synthesized as anamide during peptide synthesis leading to a molecule which isC-terminally amidated. This embodiment provides the advantage ofremoving a potential recognition point for carboxypeptidase and reducesthe potential for proteolytic degradation of the bicyclic peptide.

In one embodiment, the modified derivative comprises replacement of oneor more amino acid residues with one or more non-natural amino acidresidues. In this embodiment, non-natural amino acids may be selectedhaving isosteric/isoelectronic side chains which are neither recognisedby degradative proteases nor have any adverse effect upon targetpotency.

Alternatively, non-natural amino acids may be used having constrainedamino acid side chains, such that proteolytic hydrolysis of the nearbypeptide bond is conformationally and sterically impeded. In particular,these concern proline analogues, bulky sidechains, Cα-disubstitutedderivatives (for example, aminoisobutyric acid, Aib), and cyclo aminoacids, a simple derivative being amino-cyclopropylcarboxylic acid.

In one embodiment, the modified derivative comprises the addition of aspacer group. In a further embodiment, the modified derivative comprisesthe addition of a spacer group to the N-terminal cysteine (C_(i)) and/orthe C-terminal cysteine (C_(iii)).

In one embodiment, the modified derivative comprises replacement of oneor more oxidation sensitive amino acid residues with one or moreoxidation resistant amino acid residues. In a further embodiment, themodified derivative comprises replacement of a tryptophan residue with anaphthylalanine or alanine residue. This embodiment provides theadvantage of improving the pharmaceutical stability profile of theresultant bicyclic peptide ligand.

In one embodiment, the modified derivative comprises replacement of oneor more charged amino acid residues with one or more hydrophobic aminoacid residues. In an alternative embodiment, the modified derivativecomprises replacement of one or more hydrophobic amino acid residueswith one or more charged amino acid residues. The correct balance ofcharged versus hydrophobic amino acid residues is an importantcharacteristic of the bicyclic peptide ligands. For example, hydrophobicamino acid residues influence the degree of plasma protein binding andthus the concentration of the free available fraction in plasma, whilecharged amino acid residues (in particular arginine) may influence theinteraction of the peptide with the phospholipid membranes on cellsurfaces. The two in combination may influence half-life, volume ofdistribution and exposure of the peptide drug, and can be tailoredaccording to the clinical endpoint. In addition, the correct combinationand number of charged versus hydrophobic amino acid residues may reduceirritation at the injection site (if the peptide drug has beenadministered subcutaneously).

In one embodiment, the modified derivative comprises replacement of oneor more L-amino acid residues with one or more D-amino acid residues.This embodiment is believed to increase proteolytic stability by sterichindrance and by a propensity of D-amino acids to stabilise 8-turnconformations (Tugyi et al (2005) PNAS, 102(2), 413-418).

In one embodiment, the modified derivative comprises removal of anyamino acid residues and substitution with alanines. This embodimentprovides the advantage of removing potential proteolytic attack site(s).

It should be noted that each of the above mentioned modifications serveto deliberately improve the potency or stability of the peptide. Furtherpotency improvements based on modifications may be achieved through thefollowing mechanisms:

Incorporating hydrophobic moieties that exploit the hydrophobic effectand lead to lower off rates, such that higher affinities are achieved;

-   -   Incorporating charged groups that exploit long-range ionic        interactions, leading to faster on rates and to higher        affinities (see for example Schreiber et al, Rapid,        electrostatically assisted association of proteins (1996),        Nature Struct. Biol. 3, 427-31); and    -   Incorporating additional constraint into the peptide, by for        example constraining side chains of amino acids correctly such        that loss in entropy is minimal upon target binding,        constraining the torsional angles of the backbone such that loss        in entropy is minimal upon target binding and introducing        additional cyclisations in the molecule for identical reasons.        (for reviews see Gentilucci et al, Curr. Pharmaceutical Design,        (2010), 16, 3185-203, and Nestor et al, Curr. Medicinal Chem        (2009), 16, 4399-418).        Isotopic Variations

The present invention includes all pharmaceutically acceptable(radio)isotope-labeled peptide ligands of the invention, wherein one ormore atoms are replaced by atoms having the same atomic number, but anatomic mass or mass number different from the atomic mass or mass numberusually found in nature, and peptide ligands of the invention, whereinmetal chelating groups are attached (termed “effector”) that are capableof holding relevant (radio)isotopes, and peptide ligands of theinvention, wherein certain functional groups are covalently replacedwith relevant (radio)isotopes or isotopically labelled functionalgroups.

Examples of isotopes suitable for inclusion in the peptide ligands ofthe invention comprise isotopes of hydrogen, such as ²H (D) and ³H (T),carbon, such as ¹¹C, ¹³C and ¹⁴C, chlorine, such as ³⁶Cl, fluorine, suchas ¹⁸F, iodine, such as ¹²³I, ¹²⁵I, and ¹³¹I, nitrogen, such as ¹³N and¹⁵N, oxygen, such as ¹⁵O, ¹⁷O and ¹⁸O, phosphorus, such as ³²P, sulfur,such as ³⁵S, copper, such as ⁶⁴Cu, gallium, such as ⁶⁷Ga or ⁶⁸Ga,yttrium, such as ⁹⁰Y and lutetium, such as ¹⁷⁷Lu, and Bismuth, such as²¹³Bi.

Certain isotopically-labelled peptide ligands of the invention, forexample, those incorporating a radioactive isotope, are useful in drugand/or substrate tissue distribution studies, and to clinically assessthe presence and/or absence of the Nectin-4 target on diseased tissues.The peptide ligands of the invention can further have valuablediagnostic properties in that they can be used for detecting oridentifying the formation of a complex between a labelled compound andother molecules, peptides, proteins, enzymes or receptors. The detectingor identifying methods can use compounds that are labelled withlabelling agents such as radioisotopes, enzymes, fluorescent substances,luminous substances (for example, luminol, luminol derivatives,luciferin, aequorin and luciferase), etc. The radioactive isotopestritium, i.e. ³H (T), and carbon-14, i.e. ¹⁴C, are particularly usefulfor this purpose in view of their ease of incorporation and ready meansof detection.

Substitution with heavier isotopes such as deuterium, i.e. ²H (D), mayafford certain therapeutic advantages resulting from greater metabolicstability, for example, increased in vivo half-life or reduced dosagerequirements, and hence may be preferred in some circumstances.

Substitution with positron emitting isotopes, such as ¹¹C, ¹⁸F, ¹⁵O and¹³N, can be useful in Positron Emission Topography (PET) studies forexamining target occupancy.

Isotopically-labeled compounds of peptide ligands of the invention cangenerally be prepared by conventional techniques known to those skilledin the art or by processes analogous to those described in theaccompanying Examples using an appropriate isotopically-labeled reagentin place of the non-labeled reagent previously employed.

Synthesis

The peptides of the present invention may be manufactured syntheticallyby standard techniques followed by reaction with a molecular scaffold invitro. When this is performed, standard chemistry may be used. Thisenables the rapid large scale preparation of soluble material forfurther downstream experiments or validation. Such methods could beaccomplished using conventional chemistry such as that disclosed inTimmerman et al (supra).

Thus, the invention also relates to manufacture of polypeptides orconjugates selected as set out herein, wherein the manufacture comprisesoptional further steps as explained below. In one embodiment, thesesteps are carried out on the end product polypeptide/conjugate made bychemical synthesis.

Optionally amino acid residues in the polypeptide of interest may besubstituted when manufacturing a conjugate or complex.

Peptides can also be extended, to incorporate for example another loopand therefore introduce multiple specificities.

To extend the peptide, it may simply be extended chemically at itsN-terminus or C-terminus or within the loops using orthogonallyprotected lysines (and analogues) using standard solid phase or solutionphase chemistry. Standard (bio)conjugation techniques may be used tointroduce an activated or activatable N- or C-terminus. Alternativelyadditions may be made by fragment condensation or native chemicalligation e.g. as described in (Dawson et al. 1994. Synthesis of Proteinsby Native Chemical Ligation. Science 266:776-779), or by enzymes, forexample using subtiligase as described in (Chang et al. Proc Natl AcadSci USA. 1994 Dec. 20; 91(26):12544-8 or in Hikari et al Bioorganic &Medicinal Chemistry Letters Volume 18, Issue 22, 15 Nov. 2008, Pages6000-6003).

Alternatively, the peptides may be extended or modified by furtherconjugation through disulphide bonds. This has the additional advantageof allowing the first and second peptides to dissociate from each otheronce within the reducing environment of the cell. In this case, themolecular scaffold (e.g. TATA) could be added during the chemicalsynthesis of the first peptide so as to react with the three cysteinegroups; a further cysteine or thiol could then be appended to the N orC-terminus of the first peptide, so that this cysteine or thiol onlyreacted with a free cysteine or thiol of the second peptides, forming adisulfide-linked bicyclic peptide-peptide conjugate.

Similar techniques apply equally to the synthesis/coupling of twobicyclic and bispecific macrocycles, potentially creating atetraspecific molecule.

Furthermore, addition of other functional groups or effector groups maybe accomplished in the same manner, using appropriate chemistry,coupling at the N- or C-termini or via side chains. In one embodiment,the coupling is conducted in such a manner that it does not block theactivity of either entity.

Pharmaceutical Compositions

According to a further aspect of the invention, there is provided apharmaceutical composition comprising a peptide ligand as defined hereinin combination with one or more pharmaceutically acceptable excipients.

Generally, the present peptide ligands will be utilised in purified formtogether with pharmacologically appropriate excipients or carriers.Typically, these excipients or carriers include aqueous oralcoholic/aqueous solutions, emulsions or suspensions, including salineand/or buffered media. Parenteral vehicles include sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride and lactatedRinger's. Suitable physiologically-acceptable adjuvants, if necessary tokeep a polypeptide complex in suspension, may be chosen from thickenerssuch as carboxymethylcellulose, polyvinylpyrrolidone, gelatin andalginates.

Intravenous vehicles include fluid and nutrient replenishers andelectrolyte replenishers, such as those based on Ringer's dextrose.Preservatives and other additives, such as antimicrobials, antioxidants,chelating agents and inert gases, may also be present (Mack (1982)Remington's Pharmaceutical Sciences, 16th Edition).

The peptide ligands of the present invention may be used as separatelyadministered compositions or in conjunction with other agents. These caninclude antibodies, antibody fragments and various immunotherapeuticdrugs, such as cylcosporine, methotrexate, adriamycin or cisplatinum andimmunotoxins. Pharmaceutical compositions can include “cocktails” ofvarious cytotoxic or other agents in conjunction with the proteinligands of the present invention, or even combinations of selectedpolypeptides according to the present invention having differentspecificities, such as polypeptides selected using different targetligands, whether or not they are pooled prior to administration.

The route of administration of pharmaceutical compositions according tothe invention may be any of those commonly known to those of ordinaryskill in the art. For therapy, the peptide ligands of the invention canbe administered to any patient in accordance with standard techniques.The administration can be by any appropriate mode, includingparenterally, intravenously, intramuscularly, intraperitoneally,transdermally, via the pulmonary route, or also, appropriately, bydirect infusion with a catheter. Preferably, the pharmaceuticalcompositions according to the invention will be administered byinhalation. The dosage and frequency of administration will depend onthe age, sex and condition of the patient, concurrent administration ofother drugs, counterindications and other parameters to be taken intoaccount by the clinician.

The peptide ligands of this invention can be lyophilised for storage andreconstituted in a suitable carrier prior to use. This technique hasbeen shown to be effective and art-known lyophilisation andreconstitution techniques can be employed. It will be appreciated bythose skilled in the art that lyophilisation and reconstitution can leadto varying degrees of activity loss and that levels may have to beadjusted upward to compensate.

The compositions containing the present peptide ligands or a cocktailthereof can be administered for prophylactic and/or therapeutictreatments. In certain therapeutic applications, an adequate amount toaccomplish at least partial inhibition, suppression, modulation,killing, or some other measurable parameter, of a population of selectedcells is defined as a “therapeutically-effective dose”. Amounts neededto achieve this dosage will depend upon the severity of the disease andthe general state of the patient's own immune system, but generallyrange from 0.005 to 5.0 mg of selected peptide ligand per kilogram ofbody weight, with doses of 0.05 to 2.0 mg/kg/dose being more commonlyused. For prophylactic applications, compositions containing the presentpeptide ligands or cocktails thereof may also be administered in similaror slightly lower dosages.

A composition containing a peptide ligand according to the presentinvention may be utilised in prophylactic and therapeutic settings toaid in the alteration, inactivation, killing or removal of a selecttarget cell population in a mammal. In addition, the peptide ligandsdescribed herein may be used extracorporeally or in vitro selectively tokill, deplete or otherwise effectively remove a target cell populationfrom a heterogeneous collection of cells. Blood from a mammal may becombined extracorporeally with the selected peptide ligands whereby theundesired cells are killed or otherwise removed from the blood forreturn to the mammal in accordance with standard techniques.

Therapeutic Uses

According to a further aspect of the invention, there is provided aheterotandem bicyclic peptide complex as defined herein for use inpreventing, suppressing or treating cancer.

Examples of cancers (and their benign counterparts) which may be treated(or inhibited) include, but are not limited to tumors of epithelialorigin (adenomas and carcinomas of various types includingadenocarcinomas, squamous carcinomas, transitional cell carcinomas andother carcinomas) such as carcinomas of the bladder and urinary tract,breast, gastrointestinal tract (including the esophagus, stomach(gastric), small intestine, colon, rectum and anus), liver(hepatocellular carcinoma), gall bladder and biliary system, exocrinepancreas, kidney, lung (for example adenocarcinomas, small cell lungcarcinomas, non-small cell lung carcinomas, bronchioalveolar carcinomasand mesotheliomas), head and neck (for example cancers of the tongue,buccal cavity, larynx, pharynx, nasopharynx, tonsil, salivary glands,nasal cavity and paranasal sinuses), ovary, fallopian tubes, peritoneum,vagina, vulva, penis, cervix, myometrium, endometrium, thyroid (forexample thyroid follicular carcinoma), adrenal, prostate, skin andadnexae (for example melanoma, basal cell carcinoma, squamous cellcarcinoma, keratoacanthoma, dysplastic naevus); haematologicalmalignancies (i.e. leukemias, lymphomas) and premalignant haematologicaldisorders and disorders of borderline malignancy includinghaematological malignancies and related conditions of lymphoid lineage(for example acute lymphocytic leukemia [ALL], chronic lymphocyticleukemia [CLL], B-cell lymphomas such as diffuse large B-cell lymphoma[DLBCL], follicular lymphoma, Burkitt's lymphoma, mantle cell lymphoma,T-cell lymphomas and leukaemias, natural killer [NK] cell lymphomas,Hodgkin's lymphomas, hairy cell leukaemia, monoclonal gammopathy ofuncertain significance, plasmacytoma, multiple myeloma, andpost-transplant lymphoproliferative disorders), and haematologicalmalignancies and related conditions of myeloid lineage (for exampleacute myelogenousleukemia [AML], chronic myelogenousleukemia [CML],chronic myelomonocyticleukemia [CMML], hypereosinophilic syndrome,myeloproliferative disorders such as polycythaemia vera, essentialthrombocythaemia and primary myelofibrosis, myeloproliferative syndrome,myelodysplastic syndrome, and promyelocyticleukemia); tumors ofmesenchymal origin, for example sarcomas of soft tissue, bone orcartilage such as osteosarcomas, fibrosarcomas, chondrosarcomas,rhabdomyosarcomas, leiomyosarcomas, liposarcomas, angiosarcomas,Kaposi's sarcoma, Ewing's sarcoma, synovial sarcomas, epithelioidsarcomas, gastrointestinal stromal tumors, benign and malignanthistiocytomas, and dermatofibrosarcomaprotuberans; tumors of the centralor peripheral nervous system (for example astrocytomas, gliomas andglioblastomas, meningiomas, ependymomas, pineal tumors and schwannomas);endocrine tumors (for example pituitary tumors, adrenal tumors, isletcell tumors, parathyroid tumors, carcinoid tumors and medullarycarcinoma of the thyroid); ocular and adnexal tumors (for exampleretinoblastoma); germ cell and trophoblastic tumors (for exampleteratomas, seminomas, dysgerminomas, hydatidiform moles andchoriocarcinomas); and paediatric and embryonal tumors (for examplemedulloblastoma, neuroblastoma, Wilms tumor, and primitiveneuroectodermal tumors); or syndromes, congenital or otherwise, whichleave the patient susceptible to malignancy (for example XerodermaPigmentosum).

In a further embodiment, the cancer is selected from a hematopoieticmalignancy such as selected from: non-Hodgkin's lymphoma (NHL),Burkitt's lymphoma (BL), multiple myeloma (MM), B chronic lymphocyticleukemia (B-CLL), B and T acute lymphocytic leukemia (ALL), T celllymphoma (TCL), acute myeloid leukemia (AML), hairy cell leukemia (HCL),Hodgkin's Lymphoma (HL), and chronic myeloid leukemia (CML).

References herein to the term “prevention” involves administration ofthe protective composition prior to the induction of the disease.“Suppression” refers to administration of the composition after aninductive event, but prior to the clinical appearance of the disease.“Treatment” involves administration of the protective composition afterdisease symptoms become manifest.

Animal model systems which can be used to screen the effectiveness ofthe peptide ligands in protecting against or treating the disease areavailable. The use of animal model systems is facilitated by the presentinvention, which allows the development of polypeptide ligands which cancross react with human and animal targets, to allow the use of animalmodels.

The invention is further described below with reference to the followingexamples.

EXAMPLES

In general, the heterotandem bicyclic peptide complex of the inventionmay be prepared in accordance with the following general method:

All solvents are degassed and purged with N₂ 3 times. A solution ofBP-23825 (1.0 eq), HATU (1.2 eq) and DIEA (2.0 eq) in DMF is mixed for 5minutes, then Bicycle1 (1.2 eq.) is added. The reaction mixture isstirred at 40° C. for 16 hr. The reaction mixture is then concentratedunder reduced pressure to remove solvent and purified by prep-HPLC togive intermediate 2.

A mixture of intermediate 2 (1.0 eq) and Bicycle2 (2.0 eq) are dissolvedin t-BuOH/H₂O (1:1), and then CuSO₄ (1.0 eq), VcNa (4.0 eq), and THPTA(2.0 eq) are added. Finally, 0.2 M NH₄HCO₃ is added to adjust pH to 8.The reaction mixture is stirred at 40° C. for 16 hr under N₂ atmosphere.The reaction mixture was directly purified by prep-HPLC.

More detailed experimental for the heterotandem bicyclic peptide complexof the invention is provided herein below:

Example 1: Synthesis of BCY13272

Procedure for Preparation of BCY14964

A mixture of BP-23825 (155.5 mg, 249.40 μmol, 1.2 eq), and HATU (95.0mg, 249.92 μmol, 1.2 eq) was dissolved in NMP (1.0 mL), then the pH ofthis solution was adjusted to 8 by dropwise addition of DIEA (64.6 mg,499.83 μmol, 87.0 μL, 2.4 eq), and then the solution was allowed to stirat 25° C. for 5 min. BCY13118 (500.0 mg, 207.83 μmol, 1.0 eq) wasdissolved in NMP (5.0 mL), and then added to the reaction solution, thepH of the resulting solution was adjusted to 8 by dropwise addition ofDIEA. The reaction mixture was stirred at 25° C. for 45 min. LC-MSshowed BCY13118 was consumed completely and one main peak with desiredm/z was detected. The reaction mixture was concentrated under reducedpressure to remove solvent and produced a residue. The residue was thenpurified by preparative-HPLC to give BCY14964 (1.35 g, 403.46 μmol,64.7% yield, 90% purity) as a white solid. Calculated MW: 3011.53,observed m/z: 1506.8 ([M+2H]²⁺), 1005.0 ([M+3H]³⁺).

Procedure for Preparation of BCY13272 (Formula in FIG. 6)

A mixture of BCY8928 (644.0 mg, 290.55 μmol, 2.5 eq), THPTA (50.5 mg,116.22 μmol, 1.0 eq), CuSO₄ (0.4 M, 145.0 μL, 0.5 eq) and sodiumascorbate (82.0 mg, 464.89 μmol, 4.0 eq) were dissolved in t-BuOH/0.2 MNH₄HCO₃ (1:1, 6.0 mL). The pH of this solution was adjusted to 7.5 bydropwise addition of 0.2 M NH₄HCO₃ (in 1:1 t-BuOH/0.2 M NH₄HCO₃), andthen the solution was stirred at 25° C. for 3 min. BCY14964 (350.0 mg,116.22 μmol, 1.0 eq) was dissolved in t-BuOH/0.2 M NH₄HCO₃ (1:1, 11.0mL), and then dropped into the stirred solution. All solvents here werepre-degassed and purged with N₂. The pH of this solution was adjusted to7.5 by dropwise addition of 0.2 M NH₄HCO₃ (in 1:1 t-BuOH/0.2 M NH₄HCO₃),and the solution turned light yellow. The reaction mixture was stirredat 25° C. for 6 hr under N₂ atmosphere. LC-MS showed one main productpeak with desired m/z was detected. The reaction mixture was filteredand concentrated under reduced pressure to give a residue. The crudeproduct was purified by preparative HPLC, and BCY13272 (1.75 g, 235.01μmol, 67.40% yield, 94% purity) was obtained as a white solid.Calculated MW: 7446.64, observed m/z: 1242.0 ([M+6H]⁶⁺), 1491.0([M+5H]⁵⁺).

Example 2: Synthesis of BCY14414

Procedure for Preparation of BCY14798

A mixture of BCY14964 (55.0 mg, 18.26 μmol, 1.0 eq), BCY8928 (32.4 mg,14.61 μmol, 0.8 eq), and THPTA (39.8 mg, 91.32 μmol, 5.0 eq) wasdissolved in t-BuOH/0.2 M NH₄HCO₃ (1:1, 0.5 mL, pre-degassed and purgedwith N₂), and then CuSO₄ (0.4 M, 23.0 μL, 0.5 eq) and sodium ascorbate(72.0 mg, 365.27 μmol, 20.0 eq) were added under N₂. The pH of thissolution was adjusted to 7.5 by dropwise addition of 0.2 M NH₄HCO₃ (in1:1 t-BuOH/0.2 M NH₄HCO₃), and the solution turned to light yellow. Thereaction mixture was stirred at 25° C. for 1.5 h under N₂ atmosphere.LC-MS showed BCY14964 remained, compound BCY8928 was consumedcompletely, and one main peak with desired m/z was detected. Thereaction mixture was filtered and concentrated under reduced pressure togive a residue. The crude product was purified by preparative HPLC, andBCY14798 (51 mg, 9.17 μmol, 33.37% yield, 94% purity) was obtained as awhite solid. Calculated MW: 5229.07, observed m/z: 1308.3 ([M+4H]⁴⁺),1046.7 ([M+5H]⁵⁺).

Procedure for Preparation of BCY14414 (Formula in FIG. 7)

A mixture of BCY14798 (21.0 mg, 4.02 μmol, 1.0 eq), BCY13389 (10.0 mg,4.42 μmol, 1.1 eq), and THPTA (1.8 mg, 4.02 μmol, 1.0 eq) was dissolvedin t-BuOH/0.2 M NH₄HCO₃ (1:1, 0.5 mL, pre-degassed and purged with N₂),and then CuSO₄ (0.4 M, 5.0 μL, 0.5 eq) and sodium ascorbate (2.8 mg,16.06 μmol, 4.0 eq) were added under N₂. The pH of this solution wasadjusted to 7.5 by dropwise addition of 0.2 M NH₄HCO₃ (in 1:1 t-BuOH/0.2M NH₄HCO₃) and the solution turned to light yellow. The reaction mixturewas stirred at 25° C. for 2 hr under N₂ atmosphere. LC-MS showedBCY14798 was consumed completely, some BCY13389 remained and one mainpeak with desired m/z was detected. The reaction mixture was filteredand concentrated under reduced pressure to give a residue. The crudeproduct was purified by preparative and BCY14414 (20 mg, 2.40 μmol,59.73% yield, 90.9% purity) was obtained as a white solid. CalculatedMW: 7503.74, observed m/z: 1251.5 ([M+5H]⁵⁺), 1072.9 ([M+7H]⁷⁺).

Example 3: Synthesis of BCY14417

Procedure for Preparation of BCY14417 (Formula in FIG. 8)

A mixture of BCY14414 (13.0 mg, 1.73 μmol, 1.0 eq) and biotin-PEG12-NHSester (CAS 365441-71-0, 4.2 mg, 4.50 μmol, 2.6 eq) was dissolved in DMF(0.5 mL). The pH of this solution was adjusted to 8 by dropwise additionof DIEA. The reaction mixture was stirred at 25° C. for 0.5 hr. LC-MSshowed BCY14414 was consumed completely, and one main peak with desiredm/z was detected. The reaction mixture was filtered and concentratedunder reduced pressure to give a residue. The crude product was purifiedby preparative HPLC and BCY14417 (9.0 mg, 1.07 μmol, 80.49% yield, 90.8%purity) was obtained as a white solid. Calculated MW: 8329.74, observedm/z: 1389.6 ([M+6H]⁶⁺), 1191.9 ([M+7H]⁷⁺).

Example 4: Synthesis of BCY14418

Procedure for Preparation of BCY14418 (Formula in FIG. 9)

A mixture of BCY14414 (5.6 mg, 0.75 μmol, 1.0 eq) and Alexa Fluor® 488(0.9 mg, 1.49 μmol, 2.0 eq) was dissolved in DMF (0.3 mL). Then pH ofthis solution was adjusted to 8 by dropwise addition of DIEA. Thereaction mixture was stirred at 25° C. for 1.0 hr. LC-MS showed BCY14414was consumed completely, and one main peak with desired m/z wasdetected. The reaction mixture was filtered and concentrated underreduced pressure to give a residue. The crude product was purified bypreparative HPLC, and BCY14418 (2.3 mg, 0.25 μmol, 32.89% yield, 85.6%purity) was obtained as a red solid. Calculated MW: 8020.19, observedm/z: 1337.2 ([M+6H]⁶⁺).

Example 5: Synthesis of BCY15217

Procedure for Preparation of BCY15217 (Formula in FIG. 10)

A mixture of BCY14964 (20.0 mg, 6.64 μmol, 1.0 eq), BCY14601 (30.5 mg,13.95 μmol, 2.1 eq), and THPTA (2.9 mg, 6.64 μmol, 1.0 eq) was dissolvedin t-BuOH/0.2 M NH₄HCO₃ (1:1, 0.5 mL, pre-degassed and purged with N₂),and then CuSO₄ (0.4 M, 16.6 μL, 1.0 eq) and sodium ascorbate (4.7 mg,26.56 μmol, 4.0 eq) were added under N₂. The pH of this solution wasadjusted to 8, and the solution turned to light yellow. The reactionmixture was stirred at 25° C. for 2 hr under N₂ atmosphere. LC-MS showedBCY14964 remained, and one main peak with desired m/z was detected. Thereaction mixture was filtered and concentrated under reduced pressure togive a residue. The crude product was purified by preparative HPLC, andBCY15217 (19.7 mg, 2.41 μmol, 36.26% yield, 96.2% purity) was obtainedas a white solid. Calculated MW: 7362.5, observed m/z: 1473.5([M+5H]⁵⁺), 1228.2 ([M+6H]⁶⁺), 1052.8 ([M+7H]⁷⁺).

Example 6: Synthesis of BCY15218

Procedure for Preparation of BCY15218 (Formula in FIG. 11)

A mixture of BCY14798 (30.0 mg, 5.74 μmol, 1.0 eq), BCY14601 (15.0 mg,6.88 μmol, 1.2 eq), and THPTA (2.5 mg, 5.74 μmol, 1.0 eq) was dissolvedin t-BuOH/0.2 M NH₄HCO₃ (1:1, 0.5 mL, pre-degassed and purged with N₂),and then CuSO₄ (0.4 M, 14.0 μL, 1.0 eq) and sodium ascorbate (4.0 mg,22.95 μmol, 4.0 eq) were added under N₂. The pH of this solution wasadjusted to 7.5 by dropwise addition of 0.2 M NH₄HCO₃ (in 1:1 t-BuOH/0.2M NH₄HCO₃), and the solution turned light yellow. The reaction mixturewas stirred at 25° C. for 2 h under N₂ atmosphere. LC-MS showed BCY14798was consumed completely, BCY14601 remained, and one main peak withdesired m/z was detected. The reaction mixture was filtered andconcentrated under reduced pressure to give a residue. The crude productwas purified by preparative HPLC, and BCY15218 (22 mg, 2.67 μmol, 46.61%yield, 95.0% purity) was obtained as a white solid. Calculated MW:7404.6, observed m/z: 1234.8 ([M+61-1]⁶⁺).

Analytical Data

The heterotandem bicyclic peptide complex of the invention was analysedusing mass spectrometry and HPLC. HPLC setup was as follows:

-   -   Mobile Phase: A: 0.1% TFA in H₂O B: 0.1% TFA in ACN    -   Flow: 1.0 ml/min    -   Column: Kintex 1.7 um C18 100 A 2.1 mm*150 mm    -   Instrument: Agilent UPLC 1290

Gradients used are 30-60% B over 10 minutes and the data was generatedas follows:

HPLC Complex Analytical Data- Retention ID Mass Spectrometry Time (min)BCY13272 Calculated MW: 7102.28, observed m/z: 7.07 1776.4 [M + 4H]4+,1421.3 [M + 5H]+Biological Data1. CD137 Reporter Assay Co-Culture with Tumor Cells

Culture medium, referred to as R1 media, is prepared by adding 1% FBS toRPMI-1640 (component of Promega kit CS196005). Serial dilutions of testarticles in R1 are prepared in a sterile 96 well-plate. Add 25 μL perwell of test articles or R1 (as a background control) to designatedwells in a white cell culture plate. Tumor cells* are harvested andresuspended at a concentration of 400,000 cells/mL in R1 media. Twentyfive (25) μL/well of tumor cells are added to the white cell cultureplate. Jurkat cells (Promega kit CS196005, 0.5 mL) are thawed in thewater bath and then added to 5 ml pre-warmed R1 media. Twenty five (25)μL/well of Jurkat cells are then added to the white cell culture plate.Incubate the cells and test articles for 6 h at 37° C., 5% CO₂. At theend of 6 h, add 75 μL/well Bio-Glo™ reagent (Promega) and incubate for10 min before reading luminescence in a plate reader (Clariostar, BMG).The fold change relative to cells alone (Jurkat cells+Cell line used inco-culture) is calculated and plotted in GraphPad Prism as log(agonist)vs response to determine EC₅₀ (nM) and Fold Induction over background(Max).

The tumor cell types used in co-culture for EphA2 are A549, PC-3 andHT29.

Data presented in FIG. 1 shows that the EphA2/CD137 heterotandemBCY13272 induces strong CD137 activation in a CD137 reporter assay inthe presence of an EphA2 expressing cell line (A549) while a non-bindingcontrol molecule (BCY13626) shows no activation of CD137.

Data presented in FIG. 1 and in Table 1 below shows that BCY13272induces strong CD137 activation in a CD137 reporter assay. Theactivation is dependent on the binding of the heterotandem to both CD137and EphA2 as shown by the absence of activity of a non-binding control(BCY13626) which does not engage EphA2 or CD137.

A summary of the EC₅₀ (nM) and Fold Induction induced by BCY13272 in aCD137 reporter assay in co-culture with an EphA2 expressing tumor cellline is reported in Table 1 below:

TABLE 1 Activity of EphA2/CD137 heterotandem bicyclic peptide complexesin a CD137 reporter assay Geo mean Complex EphA2 EC50 EC50/cell ID cellline (nM) Emax line BCY13272 PC-3 0.245 44.5 0.117 0.0805 44.2 0.0898 53A549 0.1468 25.7 0.127 0.107 23.6 0.132 30.2 HT-29 0.567 36.5 0.2790.187 26 0.205 36.42. Pharmacokinetics of Heterotandem Complex BCY13272 in SD Rats

Male SD Rats were dosed with heterotandem complex BCY13272 formulated in25 mM Histidine HCl, 10% sucrose pH 7 by IV bolus or IV infusion (15minutes). Serial bleeding (about 80 μL blood/time point) was performedvia submandibular or saphenous vein at each time point. All bloodsamples were immediately transferred into prechilled microcentrifugetubes containing 2 μL K2-EDTA (0.5M) as anti-coagulant and placed on wetice. Blood samples were immediately processed for plasma bycentrifugation at approximately 4° C., 3000 g. The precipitant includinginternal standard was immediately added into the plasma, mixed well andcentrifuged at 12,000 rpm, 4° C. for 10 minutes. The supernatant wastransferred into pre-labeled polypropylene microcentrifuge tubes, andthen quick-frozen over dry ice. The samples were stored at 70° C. orbelow as needed until analysis. 7.5 μL of the supernatant samples weredirectly injected for LC-MS/MS analysis using an Orbitrap Q Exactive inpositive ion mode to determine the concentrations of analyte. Plasmaconcentration versus time data were analyzed by non-compartmentalapproaches using the Phoenix WinNonlin 6.3 software program. C0, CI,Vdss, T½, AUC(0-last), AUC(0-inf), MRT(0-last), MRT(0-inf) and graphs ofplasma concentration versus time profile were reported. Thepharmacokinetic parameters from the experiment are as shown in Table 2:

TABLE 2 Pharmacokinetic Parameters in SD Rats Dosing Vdss Clp CompoundRoute T1/2(h) (L/kg) (ml/min/kg) BCY13272 IV Inf 2.5 1.0 7.43. Pharmacokinetics of Heterotandem Complex BCY13272 in CynomolgusMonkey

Non-naïve Cynomolgus Monkeys were dosed via intravenous infusion (15 or30 min) into the cephalic vein with 1 mg/kg of heterotandem complexBCY13272 formulated in 25 mM Histidine HCl, 10% sucrose pH 7. Serialbleeding (about 1.2 ml blood/time point) was performed from a peripheralvessel from restrained, non-sedated animals at each time point into acommercially available tube containing potassium (K2) EDTA*2H₂O(0.85-1.15 mg) on wet ice and processed for plasma. Samples werecentrifuged (3,000×g for 10 minutes at 2 to 8° C.) immediately aftercollection. 0.1 mL plasma was transferred into labelled polypropylenemicro-centrifuge tubes. 5-fold of the precipitant including internalstandard 100 ng/mL Labetalol & 100 ng/mL dexamethasone & 100 ng/mLtolbutamide & 100 ng/mL Verapamil & 100 ng/mL Glyburide & 100 ng/mLCelecoxib in MeOH was immediately added into the plasma, mixed well andcentrifuged at 12,000 rpm for 10 minutes at 2 to 8° C. Samples ofsupernatant were transferred into the pre-labeled polypropylenemicrocentrifuge tubes, and frozen over dry ice. The samples were storedat −60° C. or below until LC-MS/MS analysis. An aliquot of 40 μLcalibration standard, quality control, single blank and double blanksamples were added to the 1.5 mL tube. Each sample (except the doubleblank) was quenched with 200 μL IS1 respectively (double blank samplewas quenched with 200 μL MeOH with 0.5% tritonX-100), and then themixture was vortex-mixed well (at least 15 s) with vortexer andcentrifuged for 15 min at 12000 g, 4° C. A 10 μL supernatant wasinjected for LC-MS/MS analysis using an Orbitrap Q Exactive in positiveion mode to determine the concentrations of analyte. Plasmaconcentration versus time data were analyzed by non-compartmentalapproaches using the Phoenix WinNonlin 6.3 software program. C0, CI,Vdss, T½, AUC(0-last), AUC(0-inf), MRT(0-last), MRT(0-inf) and graphs ofplasma concentration versus time profile were reported. Thepharmacokinetic parameters for BCY13272 are as shown in Table 3.

TABLE 3 Pharmacokinetic Parameters in cynomolgous monkey T_(1/2) ClpVdss Compound Route (h) (ml/min/kg) (L/kg) BCY13272 IV infusion 8.9 4.10.82 (15 min)

FIG. 2 shows the plasma concentration vs time curve of BCY13272 from a3.6 mg/kg IV infusion (15 min) in SD Rat (n=3) and a 9.2 mg/kg IVinfusion (15 min) in cynomolgus monkey (n=3). BCY13272 has a volume ofdistribution at steady state (Vdss) of 1.0 L/kg and a clearance of 7.5mL/min/kg in rats which results in a terminal half life of 2.9 h.BCY13272 has a volume of distribution at steady state (Vdss) of 0.82L/kg and a clearance of 4.1 mL/min/kg in cyno which results in aterminal half life of 8.9 h.

4. Pharmacokinetics of Heterotandem Complex BCY13272 in CD1 Mice

6 Male CD-1 mice were dosed with 15 mg/kg of heterotandem complexBCY13272 formulated in 25 mM Histidine HCl, 10% sucrose pH 7 viaintraperitoneal or intravenous administration. Serial bleeding (about 80μL blood/time point) was performed via submandibular or saphenous veinat each time point. All blood samples were immediately transferred intoprechilled microcentrifuge tubes containing 2 μL K2-EDTA (0.5M) asanti-coagulant and placed on wet ice. Blood samples were immediatelyprocessed for plasma by centrifugation at approximately 4° C., 3000 g.The precipitant including internal standard was immediately added intothe plasma, mixed well and centrifuged at 12,000 rpm, 4° C. for 10minutes. The supernatant was transferred into pre-labeled polypropylenemicrocentrifuge tubes, and then quick-frozen over dry ice. The sampleswere stored at 70° C. or below as needed until analysis. 7.5 μL of thesupernatant samples were directly injected for LC-MS/MS analysis usingan Orbitrap Q Exactive in positive ion mode to determine theconcentrations of analyte. Plasma concentration versus time data wereanalyzed by non-compartmental approaches using the Phoenix WinNonlin 6.3software program. C0, CI, Vdss, T½, AUC(0-last), AUC(0-inf),MRT(0-last), MRT(0-inf) and graphs of plasma concentration versus timeprofile were reported.

FIG. 2 shows the plasma concentration vs time curve of BCY13272 from a5.5 mg/kg IV dose in CD1 mice (n=3); the volume of distribution (Vdss)of BCY13272 is 1.1 L/kg with a Clearance of 7.5 mUmin/kg which resultsin terminal plasma half life of 2.9 h.

5. EphA2/CD137 Heterotandem Bicyclic Peptide Complex BCY13272 InducesIFN-γ Cytokine Secretion in an MC38 Co-Culture Assay

MC38 and HT1080 cell lines were cultured according to recommendedprotocols. Frozen PBMCs from healthy human donors were thawed and washedonce in room temperature PBS with benzonase, and then resuspended inRPMI supplemented with 10% heat inactivated Fetal Bovine Serum (FBS), lxPenicillin/Streptomycin, 10 mM HEPES, and 2 mM L-Glutamine (hereinreferred to as R10 medium). 100 μl of PBMCs (1,000,000 PBMCs/ml) and 100μl of tumor cells (100,000 tumor cells/ml) (Effector:Target cell ratio(E:T) 10:1) were plated in each well of a 96 well flat bottom plate forthe co-culture assay. 100 ng/ml of soluble anti-CD3 mAb (clone OKT3) wasadded to the culture on day 0 to stimulate human PBMCs. Test, controlcompounds, or vehicle controls were diluted in R10 media and 50 μL wasadded to respective wells to bring the final volume per well to 250 μL.Plates were covered with a breathable film and incubated in a humidifiedchamber at 37° C. with 5% CO₂ for two days. Supernatants were collected24 and 48 hours after stimulation, and human IFN-γ was detected byLuminex. Briefly, the standards and samples were added to a black 96well plate. Microparticle cocktail (provided in Luminex kit, R&DSystems) was added and shaken for 2 hours at room temperature. The platewas washed 3 times using a magnetic holder. Biotin cocktail was thenadded to the plate and shaken for 1 hour at RT. The plate was washed 3times using a magnetic holder. Streptavidin cocktail was added to theplate and shaken for 30 minutes at RT. The plates were washed 3 timesusing a magnetic holder, resuspended in 100 μL of wash buffer, shakenfor 2 minutes at RT, and read using the Luminex 2000. Raw data wereanalyzed using built-in Luminex software to generate standard curves andinterpolate protein concentrations, all other data analyses and graphingwere performed using Excel and Prism software. Data represents one studywith three independent donor PBMCs tested in experimental duplicates.

Data presented in FIG. 2 and in Table 4 below shows that BCY13272induces strong CD137 activation as evidenced by IFN-γ and IL-2 secretionupon CD3 stimulation. The activation is dependent on the binding of theheterotandem to both CD137 and EphA2 as evidenced by the lack ofactivity of the non-binding controls BCY12762 and BCY13692 where theCD137 and EphA2 binders respectively comprise all D-amino acid whichresult in a non-binding analog.

TABLE 4 EC50 of IL-2 cytokine secretion induced by EphA2/CD137heterotandem bicyclic complex BCY13272 in human PBMC-MC38/HT-1080co-culture assay Complex ID Cell line EC₅₀ (nM) N = BCY13272 MC38 0.79 ±0.24 5 BCY13272 HT-1080 0.55 ± 0.47 46. Anti-Tumor Activity of BCY13272 in a Syngeneic MC38 Tumor Model

6-8 week old female C57BL/6J-hCD137 mice [B-hTNFRSF9(CD137) mice;Biocytogen] were implanted subcutaneously with 1×10⁶ MC38 cells. Micewere randomized into treatment groups (n=6/cohort) when average tumorvolumes reached around 80 mm³ and were treated with vehicle (25 mMhistidine, 10% sucrose, pH7) intravenously (IV), 8 mg/kg BCY13272, 0.9mg/kg BCY13272 and 0.1 mg/kg BCY13272 IV. All treatments were giventwice a week (BIW) for 6 doses in total. Tumor growth was monitoreduntil Day 28 from treatment initiation. Complete responder animals (n=7)were followed until day 62 after treatment initiation and re-challengedwith an implantation of 2×10⁸ MC38 tumor cells and tumor growth wasmonitored for 28 days. In parallel, naïve age-matched control huCD137C57Bl/6 mice (n=5) were implanted with 2×10⁶ MC38 tumor cells monitoredfor 28 days. The results of this experiment may be seen in FIG. 3 whereit can be seen that BCY13272 leads to significant anti-tumor activitywith complete responses observed at 0.9 (2 out of 6 complete responders)and 8 mg/kg (5 out of 6 complete responders) dose levels (FIG. 3A).Unlike in naïve age-matched control huCD137 C57Bl/6 mice (tumor takerate 100%), no tumor regrowth was observed in BCY13272 completeresponder animals (FIG. 3B). These data indicate that BCY13272 hassignificant anti-tumor activity and that the BCY13272 treatment can leadinto immunogenic memory in the complete responder animals.

7. Binding of BCY13272 to EphA2 and CD137 as Measured by SPR

(a) CD137

Biacore experiments were performed to determine k_(a) (M⁻¹s⁻¹), k_(d)(s⁻¹), K_(D) (nM) values of heterotandem peptides binding to human CD137protein. Recombinant human CD137 (R&D systems) was resuspended in PBSand biotinylated using EZ-Link™ Sulfo-NHS-LC-LC-Biotin reagent (ThermoFisher) as per the manufacturer's suggested protocol. The protein wasdesalted to remove uncoupled biotin using spin columns into PBS.

For analysis of peptide binding, a Biacore T200 or a Biacore 3000instrument was used with a XanTec CMD500D chip. Streptavidin wasimmobilized on the chip using standard amine-coupling chemistry at 25°C. with HBS—N (10 mM HEPES, 0.15 M NaCl, pH 7.4) as the running buffer.Briefly, the carboxymethyl dextran surface was activated with a 7 mininjection of a 1:1 ratio of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)/0.1 M N-hydroxy succinimide (NHS) at aflow rate of 10 μl/min. For capture of streptavidin, the protein wasdiluted to 0.2 mg/ml in 10 mM sodium acetate (pH 4.5) and captured byinjecting 120 μl of onto the activated chip surface. Residual activatedgroups were blocked with a 7 min injection of 1 M ethanolamine (pH 8.5)and biotinylated CD137 captured to a level of 270-1500 RU.

Buffer was changed to PBS/0.05% Tween 20 and a dilution series of thepeptides was prepared in this buffer with a final DMSO concentration of0.5%. The top peptide concentration was 500 nM with 6 further 2-fold or3-fold dilutions. The SPR analysis was run at 25° C. at a flow rate of90 μl/min with 60 seconds association and 900 seconds dissociation.After each cycle a regeneration step (10 μl of 10 mM glycine pH 2) wasemployed. Data were corrected for DMSO excluded volume effects asneeded. All data were double-referenced for blank injections andreference surface using standard processing procedures and dataprocessing and kinetic fitting were performed using Scrubber software,version 2.0c (BioLogic Software). Data were fitted using simple 1:1binding model allowing for mass transport effects where appropriate.

(b) EphA2

Biacore experiments were performed to determine k_(a) (M⁻¹s⁻¹), k_(d)(s⁻¹), K_(D) (nM) values of BCY13272 binding to human EphA2 protein.

EphA2 were biotinylated with EZ-Link™ Sulfo-NHS-LC-Biotin for 1 hour in4 mM sodium acetate, 100 mM NaCl, pH 5.4 with a 3× molar excess ofbiotin over protein. The degree of labelling was determined using aFluorescence Biotin Quantification Kit (Thermo) after dialysis of thereaction mixture into PBS. For analysis of peptide binding, a BiacoreT200 instrument was used with a XanTec CMD500D chip. Streptavidin wasimmobilized on the chip using standard amine-coupling chemistry at 25°C. with HBS—N (10 mM HEPES, 0.15 M NaCl, pH 7.4) as the running buffer.Briefly, the carboxymethyl dextran surface was activated with a 7 mininjection of a 1:1 ratio of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)/0.1 M N-hydroxy succinimide (NHS) at aflow rate of 10 μl/min. For capture of streptavidin, the protein wasdiluted to 0.2 mg/ml in 10 mM sodium acetate (pH 4.5) and captured byinjecting 120 μl onto the activated chip surface. Residual activatedgroups were blocked with a 7 min injection of 1 M ethanolamine (pH8.5):HBS—N(1:1). Buffer was changed to PBS/0.05% Tween 20 andbiotinylated EphA2 was captured to a level of 500-1500 RU using adilution of protein to 0.2 μM in buffer. A dilution series of thepeptides was prepared in this buffer with a final DMSO concentration of0.5% with a top peptide concentration was 50 or 100 nM and 6 further2-fold dilutions. The SPR analysis was run at 25° C. at a flow rate of90 μl/min with 60 seconds association and 900-1200 seconds dissociation.Data were corrected for DMSO excluded volume effects. All data weredouble-referenced for blank injections and reference surface usingstandard processing procedures and data processing and kinetic fittingwere performed using Scrubber software, version 2.0c (BioLogicSoftware). Data were fitted using simple 1:1 binding model allowing formass transport effects where appropriate.

FIG. 5A shows the sensorgram which demonstrates that BCY13272 binds toEphA2 (human) with an affinity of 2.0 nM. FIG. 5B shows the sensorgramthat BCY13272 binds to CD137 (human) with high affinity. Due to thepresence of 2 CD137 binding bicycles in BCY13272, the off rate fromimmobilized CD137 protein is very slow and the reported K_(D) may be anoverestimation (FIG. 4B).

The invention claimed is:
 1. A heterotandem bicyclic peptide complexcomprising: (a) a first peptide ligand which binds to EphA2 and whichhas the sequence A-[HArg]-D-C_(i)[HyP]LVNPLC_(ii)LEP[d1Nal]WTC_(iii)(SEQ ID NO: 1); conjugated via an N-(acid-PEG₃)-N-bis(PEG₃-azide) linkerto (b) two second peptide ligands which bind to CD137 both of which havethe sequence Ac—C_(i)[tBuAla]PE[D-Lys(PYA)]PYC_(ii)FADPY[Nle]C_(iii)-A(SEQ ID NO: 2); or a pharmaceutically acceptable salt thereof, whereineach of said peptide ligands comprise a polypeptide comprising threereactive cysteine groups (C_(i), C_(ii) and C_(iii)), separated by twoloop sequences, and a molecular scaffold which is1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA) andwhich forms covalent bonds with the reactive cysteine groups of thepolypeptide such that two polypeptide loops are formed on the molecularscaffold; wherein Ac represents acetyl, HArg represents homoarginine,HyP represents trans-4-hydroxy-L-proline, 1Nal represents1-naphthylalanine, tBuAla represents t-butyl-alanine, PYA represents4-pentynoic acid and Nle represents norleucine.
 2. The heterotandembicyclic peptide complex according to claim 1 which is:

or a pharmaceutically acceptable salt thereof.
 3. The heterotandembicyclic peptide complex as defined in claim 1, wherein thepharmaceutically acceptable salt is selected from the free acid or thesodium, potassium, calcium, or ammonium salt.
 4. A pharmaceuticalcomposition which comprises the heterotandem bicyclic peptide complex ofclaim 1, or a pharmaceutically acceptable salt thereof, in combinationwith one or more pharmaceutically acceptable excipients.
 5. Aheterotandem bicyclic peptide complex which is:

or a pharmaceutically acceptable salt thereof.
 6. The heterotandembicyclic peptide complex of claim 5, wherein the pharmaceuticallyacceptable salt is selected from the free acid or the sodium, potassium,calcium, or ammonium salt.
 7. A pharmaceutical composition comprisingthe heterotandem bicyclic peptide complex of claim 5, or apharmaceutically acceptable salt thereof, in combination with one ormore pharmaceutically acceptable excipients.
 8. A method of binding anEphA2 receptor in a subject in need thereof, comprising administering tothe subject the heterotandem bicyclic peptide complex as defined inclaim 1, or a pharmaceutically acceptable salt thereof.
 9. The method ofclaim 8, wherein the subject has an EphA2 associated cancer.
 10. Themethod of claim 9, wherein the cancer is lung cancer.
 11. The method ofclaim 9, wherein the cancer is colon cancer.
 12. The method of claim 9,wherein the cancer is bladder cancer.
 13. The method of claim 9, whereinthe cancer is breast cancer.
 14. The method of claim 8, wherein theheterotandem bicyclic peptide complex, or a pharmaceutically acceptablesalt thereof, is administered via an intravenous administration.
 15. Amethod of binding an EphA2 receptor in a subject in need thereof,comprising administering to the subject the heterotandem bicyclicpeptide complex as defined in claim 5, or a pharmaceutically acceptablesalt thereof.
 16. The method of claim 15, wherein the subject has anEphA2 associated cancer.
 17. The method of claim 16, wherein the canceris lung cancer.
 18. The method of claim 16, wherein the cancer is coloncancer.
 19. The method of claim 16, wherein the cancer is bladdercancer.
 20. The method of claim 16, wherein the cancer is breast cancer.21. The method of claim 15, wherein the heterotandem bicyclic peptidecomplex, or a pharmaceutically acceptable salt thereof, is administeredvia an intravenous administration.